Bioreactor to obtain bioactive substances through solid-state fermentation using macromycetes fungi

ABSTRACT

Fixed bed bioreactor with natural convection and forced draught to obtain bioactive substances by solid-state fermentation (SEF) using fungi macromycetes. This bioreactor may be from turn drum with pendulum motion and natural and forced convection to a tray bioreactor with natural convection. They are used in the production of bioactive substances as crude extracts of lignocellulosic enzymes and fungal polysaccharides obtained by using mixtures of lignocellulosic materials as substrates and macromycetes fungi as inoculum, controlling pH, humidity and particle size, inoculation rate and environmental conditions during fermentation such as temperature, relative humidity and carbon dioxide and oxygen concentration.

TECHNOLOGICAL SPHERE

The present invention belongs to the branch of mechanic engineering, specifically applied to industrial microbiology. It refers to a fixed bed bioreactor with natural convection and forced draught with modifications for a rotary drum bioreactor with pendulum motion of natural and forced convection and trays bioreactor with natural convection using macromycetes fungi to obtain lignocellulosic substrates and lignocellulosic enzymes through solid-state fermentation (SEF) process under controlled conditions.

STATE OF THE ART

There are in the market bioreactors and solid-state fermentation processes to produce lignocellulosic enzymes and polysaccharides trying with different fungi and bacteria strains to quantify the potential of each biosubstance.

Fungic polysaccharides may be exo-polysaccharides and intra-polysaccharides which are obtained from submerged fermentation and solid-state fermentation (SEF). Exo-polysaccharides, those excreted to the culture medium, have been obtained by submerged fermentation processes; while the solid-state fermentation produces especially intra-polysaccharides which are in different productive bodies of the macromycetes fungi and the mycelium.

The productive bodies of fungi are obtained through the method of bag or bed culture in substrates formulated with different organic natural materials subjected to the culture conditions required for each species in order to obtain competitive yields in the market.

These have scaling problems because the difficulty in the control of variables such as heat transfers (difficult dissipation), mass transfer (heterogeneous interaction between all molecules and all directions of the substrate). These processes have proven to be efficient enzymes production up to a pilot scale.

By using the FES with filament fungi better yield has have been obtained in the production of enzymes with less problems of growth inhibition by the substrates and higher stability of the organisms to temperature and pH changes than when these same organisms have been cultured in liquid media, and less occurrence of enzymes degradation due to the presence of unwanted proteases (Hölker et al., 2004).

It is necessary the technological development around the FES, to explore the obtaining of new substances using filamentous fungi, exploring the physiological characteristics presenting during the development of fungi cells in the FES process, such as glycerol, eritritol and arabinitol accumulation which generates an induction of glucoamylases induction, may be due to temperature and activity of water under these process conditions.

The formation of secondary metabolites of a number of filamentous fungi in the FES is associated with the formation of aerial hyphas and spores at the beginning of the secondary metabolism phase and are describe only in this type of process and not in the submerged fermentation (Hölker y Lenz, 2005).

One of the most serious reasons that stop the advances in the FES processes are the design and engineering problems of standardization and scaling that limit the results reproducibility.

The management of temperature, humidity, concentration of substrate gradients increases during the process and generate adverse situation in the solid state bed processes and rotary drum reactors or other intermittent stirring equipment. Interrelationship between environmental conditions, such as oxygen concentration, humidity and temperature levels make harder the regulation of these parameters.

The fungal development under aerobe conditions in the bioreactor results in a considerable increase in heat production caused by the rapid increase of temperature. This effect is desirable in composting processes, but is totally unwanted in biotechnological processes in the bioreactors since a large part of the enzymes produced by the FES may denaturalize by the end of process.

The present invention BIOREACTORS TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI improves the temperature, humidity and oxygen concentration conditions optimal to produce bioactive substances through the Solid-state fermentation.

DESCRIPTION OF INVENTION

BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI that may vary from i) Fixed bed bioreactor with natural convection and forced draught, ii) Rotary drum bioreactor with pendulum motion and natural and forced convection, to iii) Trays bioreactor with natural convection; to product bioactive substances such as crude extracts of lignocellulosic enzymes and fungal polysaccharides obtained through a solid-state fermentation, using mixtures of lignocellulosic material and macromycetes as inoculum.

The production process of biosubstances through the solid-state fermentation uses different lignocellulosic residuals as raw material such as culture residuals and coffee post production, sugar cane, wood, fruits, vegetables, leguminous plants, oil plants, among others. For the solid-state fermentation process different physiochemical variables are considered such as carbon-nitrogen relation, minerals concentration (calcium, magnesium, iron, zinc, manganese, among others), pH, humidity and particle size; likewise the inoculation rate and environmental conditions during the fermentation as temperature, relative humidity, carbon dioxide and oxygen concentration.

DESCRIPTION OF TECHNIQUE

The bioreactor's body (FIG. 1) which is made of an stainless steel chamber (1) placed with lateral spaces in concave shape, a base diffuser (5) with dispersion partition with positive turn for air distribution and support of the substrate containers (FIG. 2), with valves to collect exudates and washing from the equipment (2, 3), a main cover (28) with a cover allowing air exchange with the outside (with stainless steel mesh and filter) (29),three spyholes to inspect the substrates incubation (4), two samplers with receptor chambers of samples isolated from the outside (8) (see detail in FIG. 5), an air diffuser (31), two air ejectors (32), air mixer (33).

Two bodies for air lethal diffusion system (FIG. 6) and (FIG. 9) (9) confronted each with the combined partition of positive turn dispersion (6) and negative (7) divided by a central partition (8) and a base diffuser (5) with partition of positive turn dispersion to distribute air when working in natural convection and addresses air flow when the bioreactor works with forced draught.

Three substrate container boxes which may be dismantled: fixed bed (10) which supports the fixed bed of substrate formulated with lignocellulosic materials and inoculated with some species of white or brown rotting macromycetes with a capacity of 10 kg each. Each box is provided with hinges with blocks (11, 12) at each side allowing dismantling the box completely and discharge the substrate block after incubation.

A temperature and relative humidity control box (FIG. 4) (13) composed by the Thermocouple selector (14), temperature monitor of six points (15), relative humidity monitor (16), ON indication light (17), general switch (18), OFF indication light (19).

Two samplers (FIG. 5) provided with handle (20), main container for samplers with contact with the environment (21), main window for samplers (22), handle for the control window for environment air entry to the bioreactor (23), control window for environment air entry to the bioreactor (24), check valve for air entry to the reactor (25), samplers general container (26), bioreactor access window (27).

The bioreactor cover (FIG. 3) (28), is provided with three spyholes (4), two samplers (8), two handles (30) and an air and CO2 supply control upper cover (29).

Two air ejectors (FIG. 7) (32) composed by two air expanders (34), an air inlet (35) and an air conduction tube (36).

An air mixer (FIG. 8) (33) composed by a fresh air inlet (37), air recirculation (38), air outlet to the reactor (39) and a tank of fresh and recirculated air (40).

Stainless steel tubing (FIG. 1) with double air expander and a mixer for air injection and recirculation, when the bioreactor works with forced draught. This piping has air supply valves and rapid coupling for an easy piping assembly and cleaning.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Bioreactor body.

FIG. 2. substrate container boxes support.

FIG. 3. Bioreactor cover.

FIG. 4. temperature and humidity control box.

FIG. 5. Sampler.

FIG. 6. Diffuser.

FIG. 7. Air ejectors.

FIG. 8. Air mixer.

FIG. 9. Air diffuser system.

FIG. 10. Process of biosubstances production by solid-state fermentation.

PREFERRED EMBODIMENT

For the optimal performance and production of biosubstances, the fixed bed bioreactor with natural convection and forced draught is composed by a bio treatment chamber with air free head of 12.5 cm as a preferred measure, three stainless Steel boxes which may be dismantled of 25 cm height, 30 cm width and 60 cm in depth as preferred measure, disposable meshes and a bed cooling central tube; two diffuser systems confronted with a combined partition of positive turn dispersion and negative and a base diffuser with positive turn dispersion partition for air distribution, an exudate accumulation and collection system.

The bioreactor cover is provided with a gasket to seal it hermetically, an upper cover with mesh and filter for air Exchange and concentration control, three spyholes for growth supervision, two samplers with hand holes to take the sample and an accessory allowing placing the sample without the material in the bioreactors has contact with the environment.

Bioreactor has six thermocouples with an outer reading port, which are placed in beds during the equipment loading; likewise it has a relative humidity recorder with scale from 5% to 100% and room temperature of the bioreactor's inner chamber with an average capacity of 30 kg of solid substrate.

The variation of the bioreactor is given by the rotary drum bioreactor with pendulum motion and natural and forced convection, which is composed by a cylinder drum or chamber containing the substrate with a spyhole, thermocouples to measure the substrate temperature, a thermo-hygrometer to measure relative humidity and room temperature in the chamber; with an speed control system and mode of pendulum and complete turn drum with a capacity of 10 kg of inoculated substrate. The tray bioreactor with natural convection has a capacity of 100 kg of inoculated substrate. It has a general chamber containing nine trays that may be dismantled with a lower support for the substrate, an air free head equivalent to the third part of the bioreactor's size and baffles allowing addressing the exudate flow to the low collector chamber thereof.

Production processes of biosubstances by solid-state fermentation with macromycetes fungi.

FIG. 10 shows a scheme process of biosubstances production by solid-state fermentation using macromycetes fungi and lignocellulosic residuals as raw material. The Figure is a process diagram illustrating how to obtain bioactive substance by solid-state fermentation in fixed bed bioreactor with natural convection and forced draught, under controlled conditions using macromycetes fungi.

All physical and chemical variables of substrates, environmental conditions and the bioreactor's selection depend directly on the species of macromycetic fungi selected, which at the same time, produce biosubstances with particular characteristics.

RESULTS OF ANALYSIS

Biosubstances obtained in the fixed bed bioreactor with convection and forced draught have been evaluated from the exudates and colonized solid material. Enzymatic activities have been determined for the colonized solid substrate and the exudate respectively: Endoglucanase (ENG) from 8 to 26 μmol/g s.s. min and from 2 and 9 μmol/mL min, Exoglucanase (EXG) from 8 to 24 μmol/g s.s. min and 1,5-12 μmol/mL. min, β-glucosidase (β-G) from 2 to 9 μmol/g s.s. min and 0,7-2,5 μmol/mL. min, Endoxilanase (ENX) from 12-25 μmol/g s.s. min and 4-9 μmol/mL.min, lignin peroxidase (LiP) 9-18 μmol/g s.s. min and 2-7 μmol/mL. min, lacase (Lc) de 12-27 μmol/g s.s. min and 5-12 μmol/mL. min, and manganese peroxidase (MnP)from 5-13 μmol/g s.s. min and 1.5-4 μmol/mL. min.

Likewise, total polysaccharides have been determined in the exudates and colonized solid substrate with macromycetes fungi from 0.03 to 0.13 g/g s.s in the colonized solid substrate and from 0.2 to 0.8 g/L in exudates. Likewise, content of N-acetyl-D-glucosamine (NAGA) has been determined in the colonized solid substrate with each species of macromycetes fungi from 0.04 and 0.13 μg/mg s.s. 

1. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI characterized because it has fixed bed with natural convection and forced draught with a capacity of 30 kg of solid substrate which varies from rotary drum bioreactor with pendulum motion and natural and forced capacity of 10 kg of solid substrate, to a trays bioreactor with natural convection with a capacity of 100 kg of solid substrate, to produce bioactive substances as crude extracts of lignocellulosic enzymes and fungal polysaccharides obtained through solid-state fermentation, using mixtures of lignocellulosic materials as substrates and macromycetes fungi as inoculum.
 2. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because the fixed bed bioreactor with natural convection and forced draught is composed by the bioreactor body which includes a stainless steel biotreatment chamber of 12.5 cm as preferred measure, placed with concave-shaped side spaces, a base diffuser with positive turn dispersion partition to distribute aire and the substrate container boxes of substrate with valves to collect the exudates and washings from the equipment, a main cover provided with a cover allowing exchanging air with the outside, three spyholes to inspect the substrates incubation, two samplers with receptor chambers of samples isolated from outside, an air diffuser, two air ejectors, and air mixer.
 3. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because it has two bodies for air lethal diffusion system confronted with a combined partition of positive turn dispersion and negative divided by a central partition and a base diffuser with positive turn dispersion partition to distribute air when working in natural convection and addressed the air flow when bioreactors works with forced draught.
 4. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because it has three boxes which may be dismantled of 25 cm height, 30 cm width and 60 cm in depth as a preferred measure, containers for substrate formulated with lignocellulosic materials and inoculated with fungi macromycetes, each box has hinges with blocks at each side allowing that box is dismantled completely and discharge the substrate box after the incubation.
 5. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because it has a temperature and relative humidity check box (Thermocouple selector, temperature monitor of six points, relative humidity monitor, ON indication light, general switch and OFF indication light).
 6. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because it has two samplers with handle, a main container so that samplers contact the environment, a main window for samplers, control window handle for environment air entry to the bioreactor, control window for environment air entry to the bioreactor, check valve for air entry to the bioreactor, samplers general container, and bioreactor access window.
 7. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because bioreactor cover has three spyholes, two samplers, two handles and an air and CO2 supply control upper cover.
 8. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because it has two air ejectors with two air expanders, an air inlet and an air conduction tube.
 9. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because it has an air mixer with a fresh air inlet—air recirculation—air outlet to the reactor and a mixture tank for fresh and recirculated air.
 10. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 1 characterized because the piping is a stainless Steel piping with double air expander and mixer for air injection and recirculation, when the bioreactor works with forced draught. This piping has air supply valves and rapid couples for an easy piping assembly and cleaning.
 11. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI characterized because it is equipment increasing colonization speed and production of macromycetes fungi exudate with a white color and brown color rotting.
 12. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 11 characterized because it increases the colonization speed and production of basidiomycetes fungi exudate with a white rotting, Ganoderma lucidum in 68% over the base substrate of mixtures of lignocellulosic materials supplemented with corn bran, rice or wheat, calcium carbonate and calcium sulfate with a colonization speed increased from 0.67 kg of colonized substrate/day to 2.14 kg of colonized substrate/day, and the production of exudate as crude extract of lignocellulosic enzymes and fungal polysaccharides increasing from 10% (p/p)to 15% (p/p), as an exudate mass produced by the mass of wet substrate upon inoculation.
 13. BIOREACTOR TO OBTAIN BIOACTIVE SUBSTANCES THROUGH SOLID-STATE FERMENTATION USING MACROMYCETES FUNGI according to claim 11 characterized because it increase the colonization speed and production of the basidiomycetes fungi exudate with a white rotting, Coriolus versicolor in 72% over the lignocellulosic materials substrate, with an increased colonization from 0.75 kg of colonized substrate/day to 2.0 kg of colonized substrate/day, and exudate production as crude extract of lignocellulosic enzymes and fungal polysaccharides increases from 7% (p/p) to 16.7% (p/p), as an exudate mass produced by the mass of wet substrate upon inoculation. 